Leiomyomas disrupt uterine function and cause recurrent pregnancy loss, excessive uterine bleeding, and anemia in 15-30% of reproductive-age women. There are few medical treatments available for leiomyomas, and many women opt to undergo hysterectomy. Understanding how leiomyomas develop is essential for identifying new non-surgical treatments. Preliminary data suggest that the clonal expansion of a defined stem/progenitor cell population is responsible for leiomyoma growth. Employing antibody-based sorting and in vivo tumor reconstruction, we revealed 3 distinct cell populations within a leiomyoma: 1-tumor progenitor CD34+/CD49b+ cells (5%) with self-renewing capacity, which express stem cell markers and the cytokine receptor RANK but are deficient in estrogen and progesterone (E+P) receptors (ER?/PR); 2-intermediately differentiated CD34+/CD49b- ?support? cells (7%) with lower tumorigenicity and high levels of ER?/PR and the cytokine RANKL; and 3-fully differentiated CD34-/CD49b- cells (88%) with negligible tumorigenic potential and high ER?/PR levels. Only the CD34+/CD49b+ cell population was indispensable for robust tumor formation in response to E+P in vivo. These cells, however, are ER?/PR deficient, suggestive of a paracrine mechanism. Our long-range objective is to define the molecular interactions between distinct uterine leiomyoma cell populations that are responsible for self-renewal, proliferation, and E+P responsiveness. The overall hypothesis is that a small stem cell population, devoid of ER? or PR, is essential for E+P-dependent growth, and that steroid-initiated alterations in ER?/PR-expressing support cells are transduced to the stem cells by RANKL/RANK signals. Microarray expression analysis suggested a hierarchical differentiation pattern: CD34+/CD49b+? CD34+/CD49b-? CD34-/CD49b- and multiple paracrine interactions between the CD34+/CD49b+ and CD34+/CD49b- populations, with the latter acting as support cells. E+P induced RANKL expression by over 100-fold in leiomyoma tissue. RANKL induced Cyclin D1 and BCL2 expression and expanded the CD34+/CD49b+ stem cell population. RANKL also activated ERK and NF?B in leiomyoma cells. Using xenografts of sorted leiomyoma cell populations under the mouse kidney capsule and cultures of tissue explants, we will test our hypothesis in vivo and in vitro in the following Aims: 1-Determine whether a small but distinct leiomyoma stem cell population serves as tumor progenitors. We will test the hypothesis that CD34+/CD49b+ cells are responsible for self-renewal, proliferation and robust tumor growth. 2-Define the biological roles of the RANKL/RANK pathway in leiomyoma progenitor/stem cell function. We will test the hypothesis that RANKL/RANK signaling acts in response to E+P and mediates self-renewal and proliferation of CD34+/CD49b+ tumor-initiating cells. In summary, our work will shift the therapeutic focus from the total leiomyoma cell mass to a small stem cell population and paracrine signaling. We expect to identify new therapeutic targets to help prevent tumorigenesis and reduce leiomyoma size and associated symptoms.